Functional analysis of the exocyst subunit BcExo70 in Botrytis cinerea.

Botrytis cinerea is one of the most important saprophytic plant pathogenic fungi. The exocyst complex and exocytosis was demonstrated to be involved in fungal development and plant infection. Here, we investigated the function of an exocyst subunit gene Bcexo70 in B. cinerea. The results show that knockout of the Bcexo70 gene significantly reduced the fungal growth and hindered the production of conidia and sclerotia. The Bcexo70 deletion strains showed a severe decrease in virulence toward tomato leaves and reduced secretion of cell wall-degrading enzyme. Confocal and electronic microscopic observation showed that the vesicles in the Bcexo70 mutants were enlarged and scattered in the cytoplasm compared to the regular distribution in the hyphal tip in wild-type strain. This study showed that the exocyst gene Bcexo70 is crucial for fungal growth, conidiation and pathogenicity in B. cinerea.

The Effect of (1S,2R-((3-bromophenethyl)amino)-N-(4-chloro-2-trifluoromethylphenyl) cyclohexane-1 -sulfonamide) on Botrytis cinerea through the Membrane Damage Mechanism.

In recent years, Botrytis cinerea has led to serious yield losses because of its resistance to fungicides. Many sulfonamides with improved properties have been used. (1S,2R-((3-bromophenethyl)amino)-N-(4-chloro-2-trifluoromethylphenyl)cyclohexane-1-sulfonamide) (abbreviation: SYAUP-CN-26) is a new sulfonamide compound that has excellent activity against B. cinerea. This study investigated the effect of SYAUP-CN-26 on electric conductivity, nucleic acids leakage, malondialdehyde (MDA) content, and reducing sugars and membrane structure reduction of B. cinerea. The results showed that the cell membrane permeability of B. cinerea increased with increasing concentrations of SYAUP-CN-26; meanwhile, the sugar content decreased, the malondialdehyde content increased, and relative electric conductivity and nucleic acid substance leakage were observed in the cell after exposure to 19.263 mg/L SYAUP-CN-26 for 24 h. After 48 h of exposure to 1.823 mg/L and 19.263 mg/L SYAUP-CN-26, the cell membranes of B. cinerea mycelia were observed to be damaged under propidium iodide (PI) and transmission electron microscopy (TEM) observations. It is assumed that SYAUP-CN-26 was responsible for the damage of cell membrane. Overall, the results indicate that SYAUP-CN-26 could inhibit the growth of B. cinerea cells by damaging the cell membranes.

Ultra-Structural Alterations in Botrytis cinerea-The Causal Agent of Gray Mold-Treated with Salt Solutions.

Potassium bicarbonate (PB), calcium chelate (CCh), and sodium silicate (SSi) have been extensively used as antifungal generally recognized as safe (GRAS) compounds against plant pathogenic fungi. In this research, in in vitro tests, the radial growth, conidial germination, and germ tube elongation of Botrytis cinerea was completely inhibited at 0.3% of PB, SSi, and CCh. In in vivo tests, application of PB, SSi, and CCh completely inhibited the occurrence of gray mold incidence of inoculated 'Italia' grape berries at concentrations of 1.0, 0.8, and 0.8%, respectively. In order to investigate the detailed mechanisms by which salts exhibited antifungal activity, we analyzed their influence on morphological changes by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and also on reactive species of oxygen (ROS), mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) content. Defects such as malformation and excessive septation were detected on salt-treated hyphae morphology observed by SEM. The internal structure of conidia treated or not with salt solutions was examined by TEM. In treated conidia, most of the conidia were affected and cellular vacuolization and cytoplasmic disorganization was observed. For ROS accumulation, a higher increase was observed in fluorescent conidia in presence of PB, SSi, and CCh by 75, 68, and 70% as compared to control, respectively. MMP was significantly decreased after salt application indicating a loss of mitochondria function. Also, luminescence showed that B. cinerea-conidia treated with salts contained less ATP than the untreated conidia. The results obtained herein are a step towards a comprehensive understanding of the mode of action by which salts act as antifungal agents against B. cinerea.

The autophagy-related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea.

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real-time polymerase chain reaction (qRT-PCR) assays showed that the expression of BcATG1 was up-regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re-introduction of an intact copy of the wild-type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.

Antifungal activity of ß-carbolines on Penicillium digitatum and Botrytis cinerea.

ß-carbolines (ßCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six ßCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic ßCs (harmine, harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-ßCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases.

Synthesis, Fungicidal Activity and Mode of Action of 4-Phenyl-6-trifluoromethyl-2-aminopyrimidines against Botrytis cinerea.

Anilinopyrimidines are the main chemical agents for management of Botrytis cinerea. However, the drug resistance in fungi against this kind of compounds is very serious. To explore new potential fungicides against B. cinerea, a series of 4-phenyl-6-trifluoromethyl-2-amino-pyrimidine compounds (compounds III-1 to III-22) were synthesized, and their structures were confirmed by ¹H-NMR, IR and MS. Most of these compounds possessed excellent fungicidal activity. The compounds III-3 and III-13 showed higher fungicidal activity than the positive control pyrimethanil on fructose gelatin agar (FGA), and compound III-3 on potato dextrose agar (PDA) indicated high activity compared to the positive control cyprodinil. In vivo greenhouse results indicated that the activity of compounds III-3, III-8, and III-11 was significantly higher than that of the fungicide pyrimethanil. Scanning electron micrography (SEM) and transmission electron micrography (TEM) were applied to illustrate the mechanism of title compounds against B. cinerea. The title compounds, especially those containing a fluorine atom at the ortho-position on the benzene ring, could maintain the antifungal activity against B. cinerea, but their mechanism of action is different from that of cyprodinil. The present study lays a good foundation for us to find more efficient reagents against B. cinerea.

Fluorescent in situ hybridization for the localization of viruses, bacteria and other microorganisms in insect and plant tissues.

Methods for the localization of cellular components such as nucleic acids, proteins, cellular vesicles and more, and the localization of microorganisms including viruses, bacteria and fungi have become an important part of any research program in biological sciences that enable the visualization of these components in fixed and live tissues without the need for complex processing steps. The rapid development of microscopy tools and technologies as well as related fluorescent markers and fluorophores for many cellular components, and the ability to design DNA and RNA sequence-based molecular probes and antibodies which can be visualized fluorescently, have rapidly advanced this field. This review will focus on some of the localizations methods which have been used in plants and insect pests in agriculture, and other microorganisms, which are rapidly advancing the research in agriculture-related fields.

Function of small GTPase Rho3 in regulating growth, conidiation and virulence of Botrytis cinerea.

Small GTPases of the Rho family play an important role in regulating biological processes in fungi. In this study, we mainly investigated the biological functions of Rho3 in Botrytis cinerea, and found that deletion of the rho3 from B. cinerea significantly suppressed vegetative growth and conidiation, reduced appressorium formation and decreased virulence. Microscopy analysis revealed that the distance between septa was increased in the Δrho3 mutant. In addition, mitochondria were suggested to be the main sources of intracellular reactive oxygen species (ROS) in B. cinerea based on dual staining with 2',7'-dichlorodihydrofluorescein diacetate and MitoTracker orange. The Δrho3 mutant showed less accumulation of ROS in the hyphae tips compared to the WT strain of B. cinerea. These results provide the novel evidence to ascertain the function of small GTPase Rho3 in regulating growth, conidiation and virulence of B. cinerea.

The possible mechanism of antifungal action of tea tree oil on Botrytis cinerea.

AIMS: Tea tree oil (TTO) has been confirmed in previous study as a potential natural antifungal agent to control Botrytis cinerea and grey mould in fresh fruit. However, the mechanism of its action has not been clearly revealed, and some hypotheses mainly depended on the results obtained from the bacterial test. For the antifungal mechanism, the effect of TTO on the mycelium morphology and ultrastructure, cell wall and membrane, and membrane fatty acid composition of B. cinerea was investigated in vitro experiments. METHODS AND RESULTS: Tea tree oil in vapour or contact phase exhibited higher activity against the mycelial growth of B. cinerea. Observations using scanning electron microscope and transmission electron microscope revealed that the mycelial morphology and ultrastructure alternations caused by TTO are the markedly shriveled or flatted empty hyphae, with thick cell walls, ruptured plasmalemma and cytoplasmic coagulation or leakage. Furthermore, TTO caused significantly higher alkaline phosphatase activity after 4-h treatment and markedly higher absorbance at 260 nm and electric conductivity in the external hyphae of fungi after 16-h treatment. Moreover, decreased unsaturated/saturated fatty acid ratio of the fungal membrane was also observed after TTO treatment. CONCLUSIONS: The methodology used in this study confirmed that the cell wall destroyed firstly in the presence of TTO, and then the membrane fatty acid composition changed, which resulted in the increasing of membrane permeability and releasing of cellular material. The above findings may be the main reason for TTO's antifungal ability to B. cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of TTO antifungal action to B. cinerea is helpful for its commercial application on the preservation of fresh fruit and vegetables.

The epiphytic fungus Pseudozyma aphidis induces jasmonic acid- and salicylic acid/nonexpressor of PR1-independent local and systemic resistance.

Pseudozyma spp. are yeast-like fungi, classified in the Ustilaginales, which are mostly epiphytic or saprophytic and are not pathogenic to plants. Several Pseudozyma species have been reported to exhibit biological activity against powdery mildews. However, previous studies have reported that Pseudozyma aphidis, which can colonize plant surfaces, is not associated with the collapse of powdery mildew colonies. In this report, we describe a novel P. aphidis strain and study its interactions with its plant host and the plant pathogen Botrytis cinerea. This isolate was found to secrete extracellular metabolites that inhibit various fungal pathogens in vitro and significantly reduce B. cinerea infection in vivo. Moreover, P. aphidis sensitized Arabidopsis (Arabidopsis thaliana) plants' defense machinery via local and systemic induction of pathogenesis-related1 (PR1) and plant defensin1.2 (PDF1.2) expression. P. aphidis also reduced B. cinerea infection, locally and systemically, in Arabidopsis mutants impaired in jasmonic acid (JA) or salicylic acid (SA) signaling. Thus, in addition to direct inhibition, P. aphidis may inhibit B. cinerea infection via induced resistance in a manner independent of SA, JA, and Nonexpressor of PR1 (NPR1). P. aphidis primed the plant defense machinery and induced stronger activation of PDF1.2 after B. cinerea infection. Finally, P. aphidis fully or partially reconstituted PR1 and PDF1.2 expression in npr1-1 mutant and in plants with the SA hydroxylase NahG transgene, but not in a jasmonate resistant1-1 mutant, after B. cinerea infection, suggesting that P. aphidis can bypass the SA/NPR1, but not JA, pathway to activate PR genes. Thus, either partial gene activation is sufficient to induce resistance, or the resistance is not directed solely through PR1 and PDF1.2 but probably through other pathogen-resistance genes or pathways as well.

Antagonistic effects of Bacillus cereus strain B-02 on morphology, ultrastructure and cytophysiology of Botrytis cinerea.

The study on antagonistic mechanism of biocontrol strains gives the premise and basis for efficient and stable biological control. This study aimes to overcome of biocontrol agent in aspects of complicated and diversified mode of action, short-lasting and unstable efficacy in the production processes. This study elucidated the antagonistic mechanism of Bacillus cereus strain B-02 on Botrytis cinerea by detecting changes in morphology, ultrastructure and physiology in affected hyphae of Botrytis cinerea. Which provided certain theoretical and practical significance for biological control of gray mould caused by B. cinerea. B. cereus strain B-02 isolated from tomato rhizosphere mightily suppressed gray mold in tomato caused by B. cinerea. Spore germination and hyphal growth of B. cinerea were inhibited by B. cereus strain B-02. Changes of cell morphology such as distortion, shrinking and swelling were observed by SEM. TEM observation further indicated the ultrastructural alterations of hyphae, including mitochondrion reduction, un-membranous inclusion in cytoplasm, considerable thickening of cell walls, and electronic density enhancement. LSCM observation revealed the fluorescence intensity of nucleus DNA, mitochondrion DNA and reactive oxygen radical in treated hyphae were all stronger than control and the difference was significant (P < 0.01). These results indicated that the antagonistic effects of B. cereus strain B-02 on B. cinerea were likely due to a combination of abnormal synthesis of nucleus DNA and mitochondrion DNA and multifarious ultrastructural alterations in hyphal cell.

Effects of resveratrol on the ultrastructure of Botrytis cinerea conidia and biological significance in plant/pathogen interactions.

Many roles have been ascribed to stilbenes, namely as antimicrobial, deterrent or repellent compounds in plants, protecting them from attacks by fungi, bacteria, nematodes or herbivores, acting both as constitutive and active defense (phytoalexin) compounds. More recently, stilbenes (especially resveratrol and its derivatives) were acclaimed for their wondrous effects and wide range of purported healing and preventive powers as cardioprotective, antitumor, neuroprotective and antioxidant agents. Although there is a huge number of works concerning the role of resveratrol in human health, reports on the antifungal activity of this compound are still scarce. This study was thus conducted in order to investigate the toxicity of resveratrol at an ultra- structural level to dormant conidia of Botrytis cinerea, the causal microorganism for gray mold. In grapevine particularly, this disease can affect all the green organs but is particularly damaging for ripening berries. Observations using transmission electron microscopy showed the occurrence of damages on conidia treated with sub-lethal doses, that is, 60µg/mL (2.6×10(-4)M) of resveratrol, a concentration usually reached in grapevine leaves and grape berries challenged by this pathogen. These results provide further data about the overall mode of action of this phytoalexin and its role in the B. cinerea/grapevine interaction.

The Botrytis cinerea Reg1 protein, a putative transcriptional regulator, is required for pathogenicity, conidiogenesis, and the production of secondary metabolites.

Botrytis cinerea, which causes gray-mold rot, attacks a wide range of plant species. To understand the infection process, the role of a putative transcriptional regulator, BcReg1 (regulator 1), in pathogenicity was studied. This transcriptional regulator shows similarity to the morphological switch regulators Candida albicans Wor1 and Histoplasma capsulatum Ryp1. Gene knock-out and complementation studies revealed that bcreg1 is required for pathogenicity. The bcreg1 mutant is able to penetrate plant tissue but is not able to cause necrotic lesions. In addition, the mutant is blocked in conidia formation and does not produce detectable levels of the sesquiterpene botrydial and the polyketide botcinic acid. Based on transcript expression levels, it can be concluded that bcreg1 is a downstream target of two mitogen-activated protein kinases, BcSak1 and Bmp3.

In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea.

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating fungal pathogen Botrytis cinerea, the causal agent of grey mould disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants, which belong to the Lamiacea family such as origanum (Origanum syriacum L. var. bevanii), lavender (Lavandula stoechas L. var. stoechas) and rosemary (Rosmarinus officinalis L.), were investigated against B. cinerea. Contact and volatile phase effects of different concentrations of the essential oils were found to inhibit the growth of B. cinerea in a dose-dependent manner. Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. A volatile vapour of origanum oil at 0.2 µg/ml air was found to completely inhibit the growth of B. cinerea. Complete growth inhibition of pathogen by essential oil of lavender and rosemary was, however, observed at 1.6 µg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, origanum oil at 12.8 µg/ml was found to inhibit the growth of B. cinerea completely. Essential oils of rosemary and lavender were inhibitory at relatively higher concentrations (25.6 µg/ml). Spore germination and germ tube elongation were also inhibited by the essential oils tested. Light and scanning electron microscopic (SEM) observations revealed that the essential oils cause considerable morphological degenerations of the fungal hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage and loss of conidiation. In vivo assays with the origanum essential oil, being the most efficient essential oil, under greenhouse conditions using susceptible tomato plants resulted in good protection against grey mould severity especially as a curative treatment. This study has demonstrated that the essential oils are potential and promising antifungal agents which could be used as biofungicide in the protection of tomato against B. cinerea.

Propamidine decreas mitochondrial complex III activity of Botrytis cinerea.

Propamidine, an aromatic diamidine compound, is widely used as an antimicrobial agent. To uncover its mechanism on pathogenetic fungi, Botrytis cinerea as an object was used to investigate effects of propamidine in this paper. The transmission electron microscope results showed that the mitochondrial membranes were collapsed after propamidine treatment, followed that mitochondria were disrupted. Inhibition of whole-cell and mitochondrial respiration by propamidine suggested that Propamidine is most likely an inhibitor of electron transport within Botrytis cinerea mitochondria. Furthermore, the mitochondrial complex III activity were inhibited by propamidine.

Genome characterization of a debilitation-associated mitovirus infecting the phytopathogenic fungus Botrytis cinerea.

The full-length sequences of Botrytiscinereamitovirus 1 (BcMV1) and an associated RNA (BcMV1-S) in strain CanBc-1c-78 of Botrytis cinerea were determined. Sequence analysis showed that BcMV1 is 2804 nt long and AU-rich (66.8%). BcMV1 shares 95% nucleotide sequence identity with Ophiostomanovo-ulmimitovirus 3b (OnuMV3b). However, it is 472 nt longer than OnuMV3b. Mitochondrial codon usage revealed that BcMV1 contains one open reading frame encoding RdRp, which is 96% identical to the RdRp of OnuMV3b. These findings confirm that BcMV1 belongs to the genus Mitovirus and is a strain of OnuMV3b. BcMV1-S is 2171 nt long and derived from BcMV1 through a single internal in-frame deletion of 633 nt, suggesting that it is a defective RNA of BcMV1. BcMV1-S was found to suppress the replication of BcMV1 and to be co-transmissible with BcMV1 through hyphal anastomosis. Its presence, however, did not alleviate the BcMV1-associated debilitation phenotypes of B. cinerea.

A new mycoparasite, Pythium lycopersicum, isolated in Isparta, Turkey: morphology, molecular characteristics, and its antagonism with phytopathogenic fungi.

Pythium lycopersicum sp. nov. has been isolated from soil samples taken in an agricultural land in the Isparta region of Southern Turkey. This oomycete is characterized by its contiguous sporangia having globose to elongated elements linked with hyphal filaments, ornamented oogonia, and monoclinous antheridia with large antheridial cells. The oomycete is reminiscent of Pythium ornamentatum described by the corresponding author in 1987 from soil samples taken in Algeria. Sequence analyses of the internal transcribed spacer (ITS) regions of rRNA show a close relationship with Pythium oligandrum and other mycoparasites possessing ornamented oogonia. Morphological and molecular features of this isolate justify its description as a new species: P. lycopersicum. When grown together with Botrytis cinerea, this oomycete shows a pronounced antagonism and suppresses the phytopathogen. The morphological details, together with the analysis of the ITS region of its rRNA, and its antagonism with some phytopathogens are discussed in this article.

Effect of cultivation conditions on spore production from Bacillus amyloliquefaciens B128 and its antagonism to Botrytis elliptica.

AIMS: To maximize spore production by Bacillus amyloliquefaciens B128, and its antagonism to the fungal pathogen Botrytis elliptica B061. METHODS AND RESULTS: In the 5-l stirred-tank bioreactor (STR), with the 0.5 vvm aeration rate, an agitation rate of 200 rev min(-1) significantly enhanced the spore yield compared to the same in 300 rev min(-1) cultivations. In a 20-l airlift bioreactor (ALR) the maximal spore production was further increased with a controlled aeration rate of 2.5 vvm operated in a 24-mesh net-draft tube mode, and no pH control cultivation. This spore yield in the 20-l ALR was five- and eightfold higher; in addition the cultivation period was 19 h shorter, compared to that obtained from shaker flask and in the 5-l STR cultivations respectively. CONCLUSIONS: Although culture conditions are still to be optimized, by using an ALR with net-draft tube, a scaling up from shaker flasks and STR to ALR of spore production by the strain B128 is technically feasible. SIGNIFICANCE AND IMPACT OF THE STUDY: The spore yields obtained using bioreactors were much higher than those previously reported. The freshly produced spore preparations from the B128 strain significantly antagonized the grey mould pathogen B. elliptica.

Licensed to kill: the lifestyle of a necrotrophic plant pathogen.

Necrotrophic plant pathogens have received an increasing amount of attention over the past decade. Initially considered to invade their hosts in a rather unsophisticated manner, necrotrophs are now known to use subtle mechanisms to subdue host plants. The gray mould pathogen Botrytis cinerea is one of the most comprehensively studied necrotrophic fungal plant pathogens. The genome sequences of two strains have been determined. Targeted mutagenesis studies are unraveling the roles played in the infection process by a variety of B. cinerea genes that are required for penetration, host cell killing, plant tissue decomposition or signaling. Our increasing understanding of the tools used by a necrotrophic fungal pathogen to invade plants will be instrumental to designing rational strategies for disease control.

Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L.

The essential oil extracted by steam distillation from the capitula of Indian Tagetes patula, Asteraceae, was evaluated for its antifungal properties and analyzed by gas chromatography and gas chromatography-mass spectrometry. Thirty compounds were identified, representing 89.1% of the total detected. The main components were piperitone (24.74%), piperitenone (22.93%), terpinolene (7.8%), dihydro tagetone (4.91%), cis-tagetone (4.62%), limonene (4.52%), and allo-ocimene (3.66%). The oil exerted a good antifungal activity against two phytopathogenic fungi, Botrytis cinerea and Penicillium digitatum, providing complete growth inhibition at 10 microl/ml and 1.25 microl/ml, respectively. The contribution of the two main compounds, piperitone and piperitenone, to the antifungal efficacy was also evaluated and ultrastructural modifications in mycelia were observed via electron microscopy, evidencing large alterations in hyphal morphology and a multisite mechanism of action.